Colonization Ability Of Pseudomonas Protegens FD6 And Functional Identification Of CbrA/B Two-Component Regulation System

Numerous strains of Pseudomonas have been identified as beneficial to plants due to their ability to function as Plant Growth-Promoting Rhizobacteria（PGPR）.These Pseudomonas strains are known for their metabolic diversity and their capacity to protect plants from pathogens,ultimately enhancing overall plant health.The ability to compete for colonization in the rhizosphere is closely linked to growth and the aforementioned abilities.Our research group has extensively studied the biocontrol abilities of Pseudomonas protegens FD6,confirming its excellent performance as a biocontrol bacterium over the course of many years.Currently,there is limited understanding of the colonization characteristics of antibacterial metabolites due to a lack of in-depth research on their synthesis and regulation during the early stages of development.In this study the colonization ability of strain FD6 was evaluated through pot experiments.The results revealed that FD6 successfully established colonization in the roots and root zones of wheat,tomato,and cucumber plants,with the highest colonization observed in cucumber roots,followed by tomato and wheat.Moreover,the amount of colonization in the root tips of tomato and cucumber was notably greater than that observed in the rhizosphere soil.No significant differences were observed in the colonization of wheat and tomato roots between the two inoculation methods of root irrigation and seed soaking.The amount of tomato rhizosphere colonization was notably greater when root irrigation was used compared to soaking seeds,with colonization reaching 105 CFU/g.In addition,FD6 demonstrates a remarkable ability.to colonize various sections of the root,including the root base,root middle,and root tip,with particularly high colonization levels observed at the root tip compared to the root base.To summarize,FD6 demonstrates a robust capacity for colonization and effectively establishes itself in the root systems of diverse plant species.The colonization capacity of rhizobacteria is typically influenced by a complex interplay of multiple genes.Researchers identified 88 conserved genes associated with colonization in the genome of Pseudomonas protegens FD6,as reported in the literature.The primary functions of these genes are related to amino acid metabolism,transport,and carbohydrate metabolism.The CbrA/B two-component regulatory system primarily regulates the metabolism of carbon and nitrogen sources in bacteria,and it may also impact bacterial colonization abilities.Therefore,this paper provides a comprehensive analysis of the role of CbrA/B in the biocontrol bacteria FD6,delving into their significance and functions in detail.The defense mechanism against Pseudomonas protegens FD6 can result in the production of diverse secondary metabolites.The control effect of FD6 and its derivative strains on tomato bacterial wilt was detected through a greenhouse pot experiment with commercial thiazole zinc as a positive control.The results of the study demonstrated that FD6 exhibited a control effect on bacterial wilt that was comparable to chemical agents.Specifically,the control effect of FD6 reached 78.86%two weeks after inoculation with Ralstonia solanacearum.However,the control effect decreased significantly after mutations in cbrA and cbrB genes.The research was conducted using test-tube plantlets as the experimental system,and it was observed that when cbrA,cbrB,and cbrAB were double-mutated,the colonization ability of the three mutants in tomato roots was affected.However,this colonization ability could be partially restored by replenishing bacteria.After seven days of inoculation,the competitive colonization ability of the three mutants in tomato roots exhibited a significant decrease,with only half of the population size observed in the wild-type.The role of cbrA and cbrB in the colonization and competitive colonization ability of FD6 is evident through their positive regulatory effects.The study of colonization-related biological phenotypes revealed that mutations in cbrA and cbrB genes led to reduced motility and increased biofilm formation.In our experiments,we investigated the impact of mutations in cbrA and cbrB genes on antibiotic synthesis,specifically Pyoluteorin（PLT）and 2,4-diacetyphloroglucinol,as revealed by the analysis of HPLC data.Performing qRT-PCR to detect the expression levels of 2,4-diacetylphloroglucinol（2,4-DAPG or PHL）and pyocyanin synthesis regulatory genes in both deletion and wild-type strains,the findings revealed a significant decrease in the expression levels of PLT and 2,4-DAPG synthesis genes pltA and phlA upon deletion of cbrA and cbrB.The activity of the transcriptional fusion structure βgalactosidase was measured,and the results provided further confirmation that CbrA/B positively regulates the transcriptional.synthesis of these two antibiotics.CbrB is a transcriptional activator protein that relies on σ54 RNA polymerase and belongs to the NtrC family of response regulators.It exhibits specific binding to 2 to 3 non-palindromic sequences that are spaced 12-16 nucleotides apart.By utilizing the conserved functional domains as reported in the literature,a comprehensive search was conducted on the FD6 genome to identify target genes potentially bound by CbrB.This investigation revealed three genes,including flgB which is implicated in flagella assembly and may be associated with motility,as well as algD a gene involved in biofilm formation and the site-specific recombinase-related gene DWF74₁6010.The promoter regions of the genes flgB,algD,and DWF74₁6010 were confirmed to be bound by CbrB through bacterial one-hybrid tests,indicating that CbrB has the ability to interact with the promoter regions of these three genes.Using pET22b（+）as the prokaryotic expression vector and BL21 as the host strain,the CbrB recombinant protein fused with the His tag protein was successfully expressed.The majority of the protein was found in the soluble form,with a molecular weight of about 52 kDa.Upon elution with 200 mM imidazole concentration eluent,high-purity CbrB protein was obtained,and the concentrated protein concentration was measured to be 0.546 mg/mL.This purified protein can now be utilized for subsequent EMSA（Electrophoretic Mobility Shift Assay）verification experiments.In conclusion,the CbrA/B regulatory system in Pseudomonas protegens FD6 exerts pleiotropic effects on various bacterial phenotypes,including colonization,motility,antibiotic synthesis,and biofilm formation.CbrB,a transcriptional regulator,interacts with the promoter regions of flgB,algD,and DWF74₁6010 genes,influencing the colonization ability of bacteria.However,further research is required to elucidate the precise regulatory mechanism of these three genes in relation to colonization in P.protegens FD6.